RESUMEN

The solution chemical properties, superoxide dismutase and catecholase activity of the copper(ii)-Ac-His-His-Gly-His-OH (hhgh) complexes were studied to identify functional and structural models of copper-containing oxidases. The solution speciation was determined in the pH range 3-11 by two independent methods (potentiometry and pH-dependent EPR measurements). The results obtained by the two methods agree very well with each other and show the formation of differently protonated CuH(x)L complexes (where x= 2 ,1, 0, -1, -2, -3) in aqueous solution. The spectroscopic (UV-Vis, CD, EPR) data indicate that the coordination of the imidazole rings is a determinant factor in all these complexes. Amide coordinated complexes are dominant only above pH 8. This offers excellent possibilities for structural/functional modelling of copper(ii) containing metalloenzymes. Indeed, the {3N(im)} coordinated CuL species (pH = 6-7) has efficient superoxide dismutase-like activity. The {3N(im),OH(-)} coordinated CuH(-1)L possesses outstanding activity to catalyze the oxidation of 3,5-di-tert-butylcatechol (H(2)dtbc) by dioxygen in 86 wt% methanol-water, providing the first example that copper(ii)-peptide complexes are able to mimic copper containing oxidases.

Asunto(s)

Materiales Biomiméticos/química , Cobre/química , Modelos Moleculares , Oligopéptidos/química , Oxidorreductasas/química , Catecoles/metabolismo , Electroquímica , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Soluciones

RESUMEN

Equilibrium and spectroscopic (1H, 13C NMR and 119Sn Mössbauer) studies in aqueous solution are reported for dimethyltin(IV) complexes of 2-hydroxyhippuric acid (Sal-Gly). Below pH 4, oxygen-coordinated complexes MLH and ML are formed. In the pH range 5-8.5, the species MLH(-1), predominates at any metal-to-ligand ratio. The ligand exchange of this species is slow on the NMR time scale, which allows its structural characterization by NMR spectroscopy: the coordination polyhedron around the tin atom is distorted trigonal bipyramidal, with tridentate [O-,N-,COO-] coordination of Sal-Gly, involving two equatorial methyl groups. The NMR results reveal that the main cause of the distortion of the polyhedron is the large CH3-Sn-CH3 angle of 136+/-4 degrees. The presented results supplement the data available on the dimethyltin(IV)-promoted amide deprotonation of peptides, and provide further arguments for the fundamental role of the carboxylate as an anchoring group in this process.

Asunto(s)

Hipuratos/química , Compuestos Orgánicos de Estaño/química , Electroquímica , Concentración de Iones de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Estructura Molecular , Soluciones/química , Espectroscopía de Mossbauer

RESUMEN

ATPase activities of molluscan adductor muscle myosins show both muscle and species specific differences: ATPase activity of catch muscle myosin is lower than that of phasic muscle myosin; a 4-5-fold difference exists between the activities of phasic striated muscle myosins from the bay scallop (Argopecten irradians) and sea scallop (Placopecten magellanicus). To characterize the light chains of these myosins we determined the cDNA sequences of the essential light chains and the regulatory light chains from Placopecten striated and catch muscle. The nucleotide sequences of the essential light chains from Placopecten striated and catch muscle myosins are identical and show 94% identity and 98% homology to the Argopecten essential light chain indicating that the tissue and species specific differences in ATPase activities are not due to the essential light chain. We identified three regulatory light chain isoforms, one from striated and two from catch muscle. Sequence differences were restricted to nucleotides encoding some of the N-terminal 52 amino acids. The three recombinant Placopecten regulatory light chain isoforms and the Argopecten regulatory light chain were incorporated into hybrid myosins that contained the essential light chain and heavy chain from Placopecten striated, Placopecten catch, or Argopecten striated muscle. Measurement of the ATPase activities of these hybrids indicates clearly that it is the myosin heavy chain and not the regulatory light chains that are responsible for the muscle and species specific differences in enzymatic activities. Analysis of genomic DNA indicated that these regulatory light chain isoforms are products of a single regulatory light chain gene that is alternatively spliced in the 5' region only.

Asunto(s)

Músculo Esquelético/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Datos de Secuencia Molecular , Moluscos , Miosinas/metabolismo

RESUMEN

We report here that the catch and striated adductor muscle myosin heavy-chain (MHC) isoforms of scallop (Argopecten irradians, previously Aequipecten irradians) are generated by alternative RNA splicing from a single gene. Scallop catch muscle cDNA and genomic DNA were amplified by PCR using primers based on the previously sequenced scallop striated muscle MHC cDNA. Mapping of the exon/intron borders and sequencing of a full-length catch muscle MHC in overlapping fragments revealed that the 24-kb gene encodes the MHC polypeptide in 27 exons and that four sets of tandem exon pairs are alternatively spliced into a striated and a catch MHC isoform. An additional alternative exon was identified in catch cDNA and is apparently spliced into a minor MHC isoform. The striated muscle-specific isoform is not expressed in other tissues, whereas the catch-type isoforms were also detected in various smooth muscles, but not in the striated one. Of the alternative exons, exons 5 and 6 encode part of the ATP-binding region and the 25-kDa/50-kDa proteolytic junction; exon 13 encodes part of one of the actin-binding regions and extends to the active site; exon 20 encodes the middle of the rod hinge region; exon 26 in the striated-specific sequence starts with the stop codon, whereas the catch-specific exon codes for an additional 10 residues. Differences between the alternative exons presumably determine the lower ATPase activity of smooth muscle myosin, contribute to the different structure of the striated and smooth muscle thick filaments, and may also be important for the molecular mechanism of the catch phenomenon.

Asunto(s)

Moluscos/genética , Músculo Liso/química , Músculos/química , Miosinas/química , Empalme Alternativo , Secuencia de Aminoácidos , Animales , ADN Complementario/genética , Exones , Expresión Génica , Genes , Intrones , Isoenzimas/genética , Datos de Secuencia Molecular , ARN Mensajero/genética , Alineación de Secuencia

RESUMEN

Specific Ca2+ binding and Ca2+ activation of ATPase activity in scallop myosin require a regulatory light chain (RLC) from regulated (molluscan or vertebrate smooth) myosin; hybrids containing vertebrate skeletal RLCs do not bind Ca2+ and their ATPase activity is inhibited. Chimeras between scallop and chicken skeletal RLCs restore Ca2+ sensitivity to RLC-free myosin provided that residues 81-117 are derived from scallop. Six mutants (R90M, A94K, D98P, N105K, M116Q, and G117C) were generated by replacing amino acids of the scallop RLC with the corresponding skeletal RLC residues in positions conserved in either regulated or nonregulated myosins. Ca2+ binding was abolished by a G117C and a G117A mutation; however, these mutants have a decreased affinity for the heavy chain. None of the other mutations affected RLC function. Replacement of the respective cysteine with glycine in the skeletal RLC has markedly changed the regulatory properties of the molecule. The single cysteine to glycine mutation conferred to this light chain the ability to restore Ca2+ binding and regulated ATPase activity, although Ca2+ activation of the actin-activated ATPase was lower than with scallop RLC. The presence of amino acids other than glycine at this position in vertebrate skeletal myosin RLCs may explain why these are not fully functional in the scallop system. The results are in agreement with x-ray crystallography data showing the central role of G117 in stabilizing the Ca(2+)-binding site of scallop myosin.

Asunto(s)

Contracción Muscular , Miosinas/química , Miosinas/fisiología , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Pollos , Glicina , Datos de Secuencia Molecular , Moluscos , Proteínas Recombinantes de Fusión , Alineación de Secuencia , Relación Estructura-Actividad

RESUMEN

The POU DNA-binding motif defines a conserved family of eukaryotic transcription factors involved in regulation of gene expression. This bipartite motif consists of an N-terminal POU-specific domain (POUs), a flexible linker, and a C-terminal POU-specific homeodomain (POUHD). Here we describe the solution structure of a POU-specific homeodomain. An NMR model is obtained from Oct-2, a human B-cell specific transcription factor which participates in the regulation of immunoglobulin genes. A fragment of Oct-2 containing POUHD and an adjoining linker was expressed in Escherichia coli and characterized by three-dimensional nuclear magnetic resonance (3D-NMR) spectroscopy. Complete 1H and 15N resonance assignment of the POUHD moiety is presented. The POUHD solution structure, as calculated by distance geometry and simulated annealing (DG/SA), is similar to that of canonical homeodomains. A salient difference between solution and crystal structures is observed in the C-terminal segment of alpha-helix 3 (the HTH recognition helix), which is not well ordered in solution. Because this segment presumably folds upon specific DNA binding, its flexibility in solution may reduce the intrinsic DNA affinity of POUHD in the absence of POUs.

Asunto(s)

Linfocitos B , Proteínas de Unión al ADN , Genes Homeobox , Espectroscopía de Resonancia Magnética , Factores de Transcripción/química , Secuencia de Aminoácidos , Secuencia de Bases , Cristalización , ADN/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Factor 2 de Transcripción de Unión a Octámeros , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Soluciones , Termodinámica

RESUMEN

The homeodomain defines a family of transcription factors broadly involved in the regulation of gene expression. DNA recognition, as observed in three representative complexes (Engrailed, Antennapedia, and MAT alpha 2), is mediated in the major groove by a helix-turn-helix (HTH) element and in the minor groove by an N-terminal arm. The three complexes share similar overall features, but they also exhibit significant differences in DNA interactions. Because these differences may distinguish the biological activities of different classes of homeodomains, we have investigated the contribution of the Oct-2 POU-specific homeodomain (POUHD) to the specificity of the bipartite POU motif. Comparative studies of variant protein-DNA complexes demonstrate the following. (i) Mutations in an invariant residue in the POUHD HTH (N347; residue 10 of the putative recognition alpha-helix) reduce octamer binding with the relaxation of specificity at one position (5'-ATGCAAAT). The inferred HTH side chain-base interaction, although not observed in the solution structure of the Antennapedia complex, is in accord with homologous contacts in the Engrailed and MAT alpha 2 cocrystal structures. (ii) Comparison of the DNA-binding properties of POU and POUHD demonstrates that POUs and POUHD independently regulate specificity at opposite ends of the DNA site (5'-TATGCAAAT). Both domains contact the two central bases (5'-TATGCAAAT) where coordinate binding of POUS in the major groove overrides the intrinsic specificity of POUHD in the minor groove. (iii) The differential sensitivity of POU and POUHD to 2'-deoxyinosine substitutions (a minor-groove modification) suggests that POUS binding repositions the POUHD N-terminal "arm".(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Proteínas de Unión al ADN/genética , ADN/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción/genética , Adenosina/análogos & derivados , Adenosina/química , Asparagina/genética , Asparagina/metabolismo , Secuencia de Bases , ADN/química , Análisis Mutacional de ADN , Proteínas de Unión al ADN/metabolismo , Genes Homeobox , Inosina/análogos & derivados , Inosina/química , Datos de Secuencia Molecular , Unión Proteica , Relación Estructura-Actividad , Factores de Transcripción/metabolismo

RESUMEN

Discrimination between allowed and disallowed amino acid substitutions provides a powerful method for analysis of protein structure and function. Site-directed mutagenesis allows specific hypotheses to be tested, but its systematic application to entire structural motifs is inefficient. This limitation may be overcome by genetic selection, which allows rapid scoring of thousands of randomly (or pseudorandomly) generated mutants. To facilitate structural dissection of DNA-binding proteins, we have designed two generally applicable bacterial selection systems: pPLUS selects for the ability of a protein to bind to a user-defined DNA sequence, whereas pMINUS selects against such binding. Complementary positive and negative selections allow rapid mapping of critical residues. To test and calibrate the systems, we have investigated the bipartite POU domain of the human B-cell-specific transcription factor Oct-2. (i) An invariant residue (Asn347) in the DNA-recognition helix of the POU-specific homeodomain (POUHD) was substituted by each of the 19 other possible amino acids. The mutant proteins each exhibited decreased specific DNA binding as defined in vivo by genetic selection and in vitro by gel retardation; relative affinities correlate with phenotypes in the positive and negative selection systems. An essential role for Asn347 in wild-type POUHD-DNA recognition is consistent with homologous Asn-adenine interactions in cocrystal structures of canonical homeodomains. (ii) Extension of pPLUS/pMINUS selection to the POU-specific subdomain (POUs) is demonstrated by analysis of mutations in its putative helix-turn-helix (HTH) element.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Plásmidos , Factores de Transcripción , Secuencia de Bases , Sitios de Unión , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Técnicas de Transferencia de Gen , Genes Reporteros , Humanos , Enlace de Hidrógeno , Operón Lac , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Factor 2 de Transcripción de Unión a Octámeros , Fenotipo , Estructura Secundaria de Proteína , Partícula de Reconocimiento de Señal/genética , Relación Estructura-Actividad

RESUMEN

The POU motif, conserved among a family of eukaryotic transcription factors, contains two DNA-binding domains: an N-terminal POU-specific domain (POUS) and a C-terminal homeodomain (POUHD). Surprisingly, POUS is similar in structure to the helix-turn-helix domains of bacteriophage repressor and Cro proteins. Such similarity predicts a common mechanism of DNA recognition. To test this prediction, we have studied the DNA-binding properties of the human Oct-2 POU domain by combined application of chemical synthesis and site-directed mutagenesis. The POUS footprint of DNA contacts, identified by use of modified bases, is analogous to those of bacteriophage repressor-operator complexes. Moreover, a loss-of-contact substitution in the putative POUS recognition alpha-helix leads to relaxed specificity at one position in the DNA target site. The implied side chain-base contact is identical to that of bacteriophage repressor and Cro proteins. These results establish a functional analogy between the POUS and prokaryotic helix-turn-helix elements and suggest that their DNA specificities may be governed by a shared set of rules.

Asunto(s)

Proteínas de Unión al ADN/química , Factores de Transcripción/química , Secuencia de Bases , Sitios de Unión , Secuencias Hélice-Asa-Hélice , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Factor 2 de Transcripción de Unión a Octámeros , Estructura Terciaria de Proteína , Proteínas Represoras/química , Relación Estructura-Actividad , Proteínas Virales , Proteínas Reguladoras y Accesorias Virales

RESUMEN

Muscle caldesmon is a long, thin protein molecule whose N- and C-terminal regions are separated by a central region which is not present in nonmuscle caldesmon. The three regions appear to be independent structural domains since the alpha-helical content of intact muscle and liver caldesmon is a sum of the alpha-helical contents of the component thrombic fragments over a broad temperature range. Based on circular dichroism spectra of liver and muscle caldesmon and its fragments, together with secondary structure prediction algorithms, it is estimated that the N-domain consists of a string of four to five short-to-intermediate-length alpha-helices; the central domain contains a long continuous alpha-helical stretch; and the C-domain can be divided into two subregions, the N-terminal C1-region, containing a long alpha-helix, and the C-terminal C2-region, containing only random coil. The thermal unfolding of caldesmon takes place gradually without a steep transition and the unfolding is reversible upon cooling, consistent with the known "heat resistance" of caldesmon. This "continuum-of-states" unfolding contrasts with the sharp, cooperative, two-state unfolding characteristic of many proteins. The domains of caldesmon also unfold gradually with the degree of unfolding increasing in the order C-domain < intact molecule < central domain < N-domain, suggesting that the thermal stability decreases in this order.

Asunto(s)

Proteínas de Unión a Calmodulina/química , Estructura Secundaria de Proteína , Animales , Pollos , Dicroismo Circular , Estabilidad de Medicamentos , Molleja de las Aves , Calor , Hígado , Músculo Liso , Conejos , Espectrofotometría Ultravioleta , Termodinámica

RESUMEN

Caldesmon migrates as a 140-kDa protein during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), although its true molecular mass is close to 90 kDa. Since caldesmon's high acidic residue content may be responsible for this anomaly, it was reasoned that modification of these residues, with a loss of negative charge, might restore normal electrophoretic migration. Therefore caldesmon was reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of excess ethanolamine, which results in negatively charged carboxylates being converted to neutral amides without protein cross-linking. The absence of cross-linking was shown by rotary shadow electron microscopy. In accord with expectations, modified caldesmon migrated as a 94-kDa protein when compared to standards, which were much less affected by modification. The anomalous migration of caldesmon might be due to the repulsion of negatively charged SDS by caldesmon's acidic residues. Low binding of SDS to caldesmon is consistent with the fact that SDS, up to 1%, had little or no effect on the secondary structure of caldesmon, as monitored by circular dichroism. However, other mechanisms can also explain these observations. The abnormal migration of tropomyosin and calsequestrin, both of which have a high percentage of acidic amino acids, was also "normalized" by this treatment. Thus this method might have general application for the electrophoresis of proteins which have a high acidic residue content and migrate anomalously.

Asunto(s)

Proteínas de Unión a Calmodulina/química , Electroforesis en Gel de Poliacrilamida/métodos , Etildimetilaminopropil Carbodiimida , Proteínas/química , Dodecil Sulfato de Sodio , Proteínas de Unión a Calmodulina/aislamiento & purificación , Proteínas de Unión a Calmodulina/ultraestructura , Dicroismo Circular , Guanidina , Guanidinas , Microscopía Electrónica , Peso Molecular , Unión Proteica , Proteínas/aislamiento & purificación

RESUMEN

B-cell specific regulation of immunoglobulin gene expression provides a model for the interaction of promoter and enhancer elements with eukaryotic sequence-specific DNA binding proteins. A critical element of this system, the octamer site (5'-ATGCAAAT-3'), is recognized by the B-cell transcription factor Oct-2. Octamer recognition is mediated by the POU domain, a conserved structural motif which--like the zinc finger and leucine zipper--defines a family of related transcription factors. Homologies among POU sequences suggest a bipartite structure, consisting of an N-terminal POU-specific subdomain and C-terminal variant homeodomain connected by a linker of variable length and sequence. As a first step toward a molecular understanding of the Oct-2 POU domain and its mechanism of DNA recognition, we have overexpressed in Escherichia coli the intact POU domain and subdomains as thrombin-cleavable fusion proteins and have purified these fragments to homogeneity following digestion with thrombin. Biochemical and biophysical characterization yields the following results. (i) The intact POU domain (166 residues) is monomeric and exhibits high-affinity octamer-specific DNA-binding activity. (ii) Limited proteolytic digestion demonstrates that the POU domain contains two proteolytically stable subdomains (the POU-specific subdomain and the variant homeodomain) connected by a proteolytically sensitive linker. (iii) The isolated subdomains are each monomeric and do not interact to form noncovalent heterodimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Proteínas de Unión al ADN/química , ADN/química , Factores de Transcripción , Secuencia de Aminoácidos , Linfocitos B/química , Secuencia de Bases , Sitios de Unión , Cromatografía Líquida de Alta Presión , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Estabilidad de Medicamentos , Elementos de Facilitación Genéticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Mutagénesis , Factor 2 de Transcripción de Unión a Octámeros , Fragmentos de Péptidos/metabolismo , Plásmidos , Regiones Promotoras Genéticas , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Temperatura , Termolisina/metabolismo

RESUMEN

Treatment of a solution of actin and smooth muscle caldesmon with 5,5'-dithiobis(2-nitrobenzoic acid) results in the formation of a disulfide cross-link between the C-terminal penultimate residue Cys-374 of actin and Cys-580 in caldesmon's C-terminal actin-binding region. Therefore, these 2 residues are close in the actin-caldesmon complex. Since myosin also binds to actin in the vicinity of Cys-374 and since caldesmon inhibits actomyosin ATPase activity by the reduction of myosin binding to actin, then the inhibition might be by caldesmon sterically hindering or blocking myosin's interaction with actin. [Ca2+]Calmodulin, which reverses the inhibition of the ATPase activity, decreases the yield of the cross-linked species, suggesting a weakening of the caldesmon-actin interaction in the cross-linked region. It is possible to maximally cross-link one caldesmon molecule/every three actin monomers, in the absence or presence of tropomyosin, clearly ruling out an elongated, end-to-end alignment of caldesmon on the actin filament in vitro, and raising the possibility that the N-terminal part of caldesmon projects out from the filament. Reaction of 5,5'-dithiobis(2-nitrobenzoic acid)-modified actin with caldesmon leads to the same disulfide cross-linked product between actin and caldesmon Cys-580, enabling the specific labeling of the other caldesmon cysteine, residue 153, in the N-terminal part of caldesmon with a spectroscopic probe.

Asunto(s)

Actinas/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Disulfuros/metabolismo , Animales , Pollos , Reactivos de Enlaces Cruzados , Ácido Ditionitrobenzoico/química , Electroforesis en Gel de Poliacrilamida , Colorantes Fluorescentes , Molleja de las Aves/química , Músculo Liso/química , Miosinas/metabolismo , Trombina/metabolismo

RESUMEN

Subunits of gizzard smooth muscle tropomyosin, dissociated by guanidinium chloride and reassociated by high salt dialysis, form a 1:1 mixture of the beta beta and gamma gamma homodimers (Graceffa, P. (1989) Biochemistry 28, 1282-1287). The homodimers have now been separated by anion-exchange chromatography and native gel electrophoresis, enabling us to show that the native protein is composed of more than 90% heterodimer. The in vitro equilibrium distribution of heterodimer and homodimers, at close to physiological temperature and ionic conditions, was calculated from thermal unfolding profiles of separated homodimers and heterodimer, as monitored by circular dichroism. The results, for an equal proportion of beta and gamma chains, indicate a predominant formation of heterodimer via chain dissociation and chain exchange, although the proportion of heterodimer was much less than the 90-100% found in the native protein. However, the proportion of heterodimer for actin-bound tropomyosin, determined by analyzing tropomyosin sedimented with actin, was greater than 90%, which may provide a model for assembly in vivo. The end-to-end interactions of the homodimers are about the same but are much less than that of the native heterodimer, as determined by viscometry. The greater end-to-end interaction of heterodimers may lead to stronger binding to actin compared to homodimers and thus would further shift the equilibrium between heterodimer and homodimers toward heterodimer and possibly account for the almost exclusive population of heterodimer in the presence of actin. The greater end-to-end interaction of the heterodimer may also provide a functional advantage for its preferred assembly. This study also shows that the two-step thermal unfolding of the homodimer mixture is due to the formation of heterodimer via an intermediate which is a new type of tropomyosin species which forms a gel in low salt. This tropomyosin is also present in small amounts in native tropomyosin preparations.

Asunto(s)

Músculo Liso/metabolismo , Tropomiosina/química , Actinas/metabolismo , Animales , Pollos , Cromatografía por Intercambio Iónico , Dicroismo Circular , Reactivos de Enlaces Cruzados , Electroforesis en Gel de Poliacrilamida , Conformación Proteica , Espectrofotometría Ultravioleta , Temperatura

RESUMEN

Although smooth muscle caldesmon migrates as a 140- to 150-kDa protein during sodium dodecyl sulfate-gel electrophoresis, its molecular mass is around 93 kDa as determined by sedimentation equilibrium (P. Graceffa, C-L. A. Wang, and W. F. Stafford, 1988, J. Biol. Chem. 263, 14,196-14,202). Nonmuscle caldesmon migrates during electrophoresis with a molecular mass close to 77 kDa, about half that of the muscle isoform. However, it is controversial whether the molecular weight of nonmuscle caldesmon is the same or much less than that of the muscle protein. Therefore we have now determined the molecular mass of rabbit liver caldesmon by sedimentation equilibrium and found a value of 66 +/- 2 kDa, a value much smaller than that of muscle caldesmon. This new value of the molecular weight, together with a sedimentation coefficient of 2.49 +/- 0.02 S. yields an apparent length of 53 +/- 2 nm and a diameter of 1.7 nm for the liver protein. We previously estimated a length of 74 nm and a diameter of 1.7 nm for the muscle caldesmon. We have also determined the amino acid composition of liver caldesmon and found it to be similar to that of the muscle protein. In conclusion, muscle and nonmuscle caldesmons appear to have similar overall amino acid composition and tertiary structure with the smaller nonmuscle protein having a correspondingly smaller length. The difference in molecular weight between the two caldesmons is consistent with the nonmuscle protein lacking a central peptide of the muscle isoform, as suggested by E. H. Ball, and T. Kovala, (1988, Biochemistry 27, 6093-6098).

Asunto(s)

Proteínas de Unión a Calmodulina/análisis , Hígado/análisis , Aminoácidos/análisis , Animales , Proteínas de Unión a Calmodulina/aislamiento & purificación , Peso Molecular , Conejos , Ultracentrifugación

RESUMEN

The aspartic residue (Asp-189) at the base of the substrate-binding pocket of trypsin was replaced by serine (present in a similar position in chymotrypsin) through site-directed mutagenesis. The wild-type (with Asp-189 in the mature trypsin sequence) and mutant (Ser-189) trypsinogens were expressed in Escherichia coli, purified to homogeneity, activated by enterokinase, and tested with a series of fluorogenic tetrapeptide substrates with the general formula succinyl-Ala-Ala-Pro-Xaa-AMC, where AMC is 7-amino-4-methyl-coumarin and Xaa is Lys, Arg, Tyr, Phe, Leu, or Trp. As compared to [Asp189]trypsin, the activity of [Ser189]trypsin on lysyl and arginyl substrates decreased by about 5 orders of magnitude while its Km values increased only 2- to 6-fold. In contrast, [Ser189]trypsin was 10-50 times more active on the less preferred, chymotrypsin-type substrates (tyrosyl, phenylalanyl, leucyl, and tryptophanyl). The activity of [Ser189]trypsin on lysyl substrate was about 100-fold greater at pH 10.5 than at pH 7.0, indicating that the unprotonated lysine is preferred. Assuming the reaction mechanisms of the wild-type and mutant enzymes to be the same, we calculated the changes in the transition-state energies for various enzyme-substrate pairs to reflect electrostatic and hydrogen-bond interactions. The relative binding energies (E) in the transition state are as follows: EII greater than EPP greater than EPA greater than EIP approximately equal to EIA, where I = ionic, P = nonionic but polar, and A = apolar residues in the binding pocket. These side-chain interactions become prominent during the transition of the Michaelis complex to the tetrahedral transition-state complex.

Asunto(s)

Tripsina/metabolismo , Secuencia de Aminoácidos , Animales , Ácido Aspártico , Sitios de Unión , Catálisis , Gráficos por Computador , Electroquímica , Lisina , Datos de Secuencia Molecular , Mutación , Ratas , Serina , Relación Estructura-Actividad , Especificidad por Sustrato , Termodinámica , Tripsina/genética

RESUMEN

The reactivities of lysines of actin in the actin-DNAase I complex were measured by the method of reductive methylation. The reactivities of lysines in the amino-terminal part, lysines 18, 50, 61 and 68, decreased 50%, while those of lysines 237, 283 and 290 increased about 30%, in comparison with those in G-actin, when actin was bound to DNAase I. These results are consistent with the view that the amino-terminal region of actin is the binding site for DNAase I. In conjunction with our earlier work on the reactivities of lysines in F-actin (Lu, R.C. and Szilagyi, L. (1981) Biochemistry 20, 5914-5919), these results are also consistent with the view that DNAase I binds to actin at one of the regions that is involved in polymerization.

Asunto(s)

Actinas/metabolismo , Desoxirribonucleasa I/metabolismo , Lisina/metabolismo , Desoxirribonucleasa I/antagonistas & inhibidores , Electroforesis en Gel de Poliacrilamida , Metilación , Oxidación-Reducción , Unión Proteica

RESUMEN

IgM and IgG type antibody titers and levels of serum IgG, IgA and IgM were determined in healthy young and aged subjects. The proportion of subjects of low antibacterial agglutinin titers progressively increased during the 7th and 8th decades of life. Anti-streptolysin-O titers were also shifted to the lower values in aged subjects, at least until the 8th decade of life, although subnormal values compared to the young control range were less frequent than in the case of IgM type antibodies. Anti-streptokinase values did not seem affected by age. In contrast to antibody levels, serum IgM was similar or slightly higher in old compared to young subjects. Evidence is presented that the proportion of 7 S IgM drops with aging. Both IgA and IgG levels increased through the 7th, 8th and 9th decades of life. Different class immunoglobulin levels seemed to be considerably correlated and a tendency to correlate was found between IgG type antibody and serum IgG levels. Complex investigations including quantitation of antibodies to extrinsic and intrinsic antigens and serum immunoglobulins are proposed to define the humoral immune status of aged subjects and to understand the causes as well as the diagnostic and prognostic significance of old age 'imbalances'.

Asunto(s)

Envejecimiento , Anticuerpos/análisis , Inmunoglobulinas/análisis , Adulto , Anciano , Anticuerpos Antibacterianos/análisis , Formación de Anticuerpos , Antiestreptolisina/análisis , Femenino , Humanos , Masculino , Persona de Mediana Edad , Streptococcus/inmunología

Asunto(s)

Benzotiadiazinas , Hipercalcemia/inducido químicamente , Inhibidores de los Simportadores del Cloruro de Sodio/efectos adversos , Diuréticos , Femenino , Humanos , Hipertensión/tratamiento farmacológico , Persona de Mediana Edad , Inhibidores de los Simportadores del Cloruro de Sodio/uso terapéutico

RESUMEN

A simplified procedure for the preparation of alpha-actinin from rabbit skeletal muscle is described. Use is made of the observation that actin is completely eliminated by denaturation, when myofibrils are dissolved in 0.6 M MgCl2 and thereafter MgCl2 is removed by dialysis.

Asunto(s)

Actinina/aislamiento & purificación , Proteínas Musculares/aislamiento & purificación , Músculos/análisis , Animales , Magnesio/farmacología , Desnaturalización Proteica/efectos de los fármacos , Conejos